During the last 10 to 15 years, two ionization methods have become generally accepted in the mass spectrometric analysis of biochemical polymers in proteomics, genomics or metabolomics (the examination and measurement of metabolic processes) among other areas. These methods are matrix-assisted laser desorption and ionization (MALDI), which is predominantly used for solid samples prepared on sample support plates, and electrospray ionization (ESI), which is used under atmospheric pressure on samples in solution. The electrospray method can be coupled relatively easily to separation methods for mixed components such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Laser desorption, which was previously only used under vacuum, can now also be used at atmospheric pressure, making it easier to introduce the sample. MALDI is characterized by a high sample throughput through the mass spectrometer. The analyte substances, however, must be separated, preferably by a separation method which is performed upstream.
In the meantime, a whole family of ionization methods operating at atmospheric pressure has been developed. These are covered by the abbreviation API (atmospheric pressure ionization). In addition to the electrospray method used originally, there is now a pneumatic method of spraying through concentric capillaries, which is coupled to photoionization by means of UV radiation with sufficient energy (APPI=atmospheric pressure photoionization) or coupled to a method using chemical ionization by primary ions generated by corona discharge (APCI=atmospheric pressure chemical ionization). Also included is the matrix-assisted laser desorption and ionization method at atmospheric pressure (AP MALDI) previously mentioned, where is no spraying process is involved.
All ionization methods at atmospheric pressure are characterized by the formation of an ionization cloud, which can be moved with the surrounding gas. This cloud may already contain some or all of the analyte ions or else these analyte ions may be produced by intermediate processes (such as chemical ionization, droplet drying or photoionization) after the cloud has formed. For all these methods, as many of the ions as possible must be guided from this cloud, which varies in size, to the entrance of the mass spectrometer and transferred to its vacuum system.
With the original electrospray method, a voltage of several kilovolts was applied across the end of a metal spray capillary and a counter electrode which were approximately 20 to 50 mm apart.
A polarizable liquid inside the capillary (usually water but sometimes water mixed with an organic solvent such as methanol or acetonitrile) is dielectrically polarized by the electric field at the end of the spray capillary and drawn out to form a cone, the so-called Taylor cone. At the end of this cone, the surface tension of the liquid is no longer able to resist the pulling force of the electric field, which is concentrated at this point. This causes a tiny beam of liquid to be torn off. The tiny beam of liquid breaks immediately into a spray cloud of tiny drops, which are electrically charged because the surface of the liquid is dielectrically polarized. With positive drops, the electric charge arises from protons produced by the dissociation of the spray fluid. (As is known, water is dissociated under normal conditions, pH 7, into H+ and OH− ions at 10−7 parts).
Initially, the charged droplets are rapidly accelerated from the tip by the non-homogeneous electric field but rapidly decelerated in the surrounding gas. This is a drying gas which usually consists of heated nitrogen. Here, the spray cloud appears as an ionization cloud with relatively clearly defined borders. This cloud drifts with the movement of the gas; the charged particles can be pulled out of the cloud by external electric fields.
In the hot drying gas, into which the charged particles are pulled, liquid evaporates from the droplets. It has to be assumed that it is the organic solvent which evaporates first. As the diameters of the now aqueous droplets decrease, their vapor pressure increases since the so-called coordination number of the molecules decreases at the surface. The coordination number gives the number of immediate neighbors, which determines how the surface molecules are bonded to the droplets. This determines the vapor pressure. However, if the liquid evaporates rapidly there is a danger that the droplets will freeze due to the loss of heat from evaporation so further drying will be slowed down.
If the droplets are highly charged, then the charges are driven to the surface by coulombic repulsion. With charged particles, the mutual repulsion increases the vapor pressure so that molecules such as protonated water (H3O+) are driven out. Theoretical considerations have shown that this causes the smaller particles to be ‘pinched off’ and then separated. All of these processes are greatly impeded or prevent altogether by the droplets freezing.
If there are some larger molecules in the droplet which usually can be easier charged by protonation (because of their higher proton affinity) than the molecules of the liquid (or by deprotonation if the polarity of spray voltage is reversed), then the larger molecules regularly will remain ionized after the liquid has fully evaporated. At the same time, the ionized molecules continue to migrate towards the counter electrode or towards other electrodes in their vicinity due to the electric field, by the known process of ‘ion mobility’. They can then be guided according to the shape of the electric fields and the surrounding gas flow and finally transferred to the vacuum system of a mass spectrometer through a fine aperture in the wall or through a transfer capillary.
In the electrospray ion sources which have been commercially available until now, the spray cloud is located only three to five centimeters from the entrance of the electrically attractive tip of a transfer capillary. The capillary transfers the ions, enveloped in neutral gas, into the vacuum of the mass spectrometer. Because of the short distance, not all of the droplets are completely dried. Some droplets which are not dried are pulled into the transfer capillary and, therefore, into the vacuum, while others are deposited around the entrance of the transfer capillary.
The droplets are detached from the Taylor cone at the tip of the spray capillary or from the fine liquid beam at the extremely fast rate of 105 to 108 droplets per second, depending on the supply of liquid in the capillary, so the result is usually a continuous ion beam. The supply is maintained by a very smoothly operating pump, usually a spray pump. The pumps of liquid chromatographs can be used for this purpose.
The larger molecules are usually charged not just singly but multiply during this process. The larger the molecule, the greater the average charge number, although there is regularly a wide distribution of charge numbers. As a rule of thumb, the average charge number increases by about one unit of charge per 1000 to 1500 atomic mass units. However, the charge also largely depends on the fold structure of the biopolymers. Large, denatured (unfolded) biomolecular ions with masses amounting to several ten thousands of atomic mass units can certainly carry 10 to 50 charges. In the case of peptides with five to twenty amino acids (mass range from approximately 600 to 2400 atomic mass units), most ions carry two charges and the distribution in this case ranges from singly charged ions to ions with 5 charges. The charge is usually protonation, not ionization by electron loss; in other words, it is produced by the bonding of charged hydrogen atoms H+. For this reason, the ionization greatly depends on the hydrogen-ion concentration (i.e., the pH value) of the sprayed solution.
With electrospray ion sources using metal spray capillaries, the droplets initially have a self-establishing diameter of a half to two micrometers depending on the dielectric constant, pH, viscosity, conductivity, flow rate and surface tension of the liquid. Occasionally, larger droplets are also produced. Electrospray ionization is not always stable, and sometimes there are floating states which lead to irregular droplet formation and a strongly fluctuating ion beam. With liquid flows in the range of one microliter per minute, supplying a spray gas coaxially has usually been found to be a successful method of stabilizing the spraying process (“gas-supported spraying”). All commercially manufactured electrospray ion sources operate today with gas-supported spraying (see, for example, A. C. Hirabayashi and Y. K. Hirabayashi, EP 0762 473 A2 or J. L. Bertsch et al. WO 97/28 556 AI). The spray gas which is supplied has a major effect on the shape of the ionization cloud, which has an increased circumference and length.
A stable operating mode is also dependent on the properties of the spray liquid mentioned above. Frequently, a stable spray is only possible within a relatively narrow range of these parameters. For this reason, supplying a supplementary liquid, which is admixed coaxially, has been found to be successful for chromatography microcolumns that only deliver a small stream of liquid (and also for capillary electrophoresis). The supplementary liquid is able to stabilize the spray since pH values and other parameters of the liquid can be adjusted without reference to the values of the parameters in the chromatography column. However, this also reduces the concentration of the analyte.
In order, at least, to keep the larger droplets away from the transfer capillary, not pointing the spray capillary directly towards the entrance of the mass spectrometer but blowing the spray cone past the entrance while maintaining a large angle between the spray capillary and the transfer capillary has been found to be effective (J. A. Apffel et al. U.S. Pat. No. 5,750,988). The distance is selected so that the spray cloud comes to a stop, due to friction in the surrounding gas in the extended axis of the transfer capillary, about three to five centimeters from the capillary's entrance (i.e., stopping in relation to the gas flow). The larger droplets then continue to travel due to inertia and miss the transfer capillary. The ions and the charged droplets are pulled laterally out of the spray cloud towards the transfer capillary, partly dried, captured by the suction funnel in front of the capillary entrance and pulled along by viscous entrainment (gas friction) into the capillary. In this process, the ions can be concentrated in front of the transfer capillary by applying suitable fields and exploiting the ion mobility (e.g. by concentric, semi-spherical shaped grids: E. W. Sheehan et al., US02/0 011 560 AI).
The transfer capillary is usually screened by an apertured diaphragm which is used to guide the hot drying gas and shape the electric field. The flow of drying gas is guided past the entrance of the transfer capillary to the spray cloud. The electric field between the ionization cloud, apertured diaphragm and transfer capillary guides the ions from the spray cloud, through the gas flowing in the opposite direction, to the entrance of the transfer capillary. At the same time, there is often no choice but to accept that the droplets are also pulled into the transfer capillary together with the ions. These droplets are hydrodynamically focused in the transfer capillary and reach the vacuum system. An attempt is then made at repairing the damage in the vacuum system as the ions move on (see for example, A. Mordehai and S. E. Buttrill, U.S. Pat. No. 5,818,041 and WO 97/30 469 A1).
As indicated above, today, other principles which have their merits for other classes of analyte substances are also used for the ionization instead of the electrospray. The spray can therefore produce droplets by pneumatic means alone and without an electric drawing field, in which case, they do not carry a charge. The molecules can then be ionized in the droplets or after the liquid has evaporated by reacting with the primary ions from a corona discharge. This method is called APCI (atmospheric pressure chemical ionization, see, for example, Y. Takada et al., U.S. Pat. No. 5,877,495 and Y. Takada et al., U.S. Pat. No. 6,121,608). However, the molecules can also be ionized by UV radiation with a photon energy of about seven to ten electron volts, as known from ion mobility spectrometry. This is known as APPI (atmospheric pressure photoionization).
Special versions of the electrospray method relate to apparatuses for particularly low flow rates in the spray. By using very fine capillary tips, it is possible to maintain the flow rate at a few tens of nanoliters per minute. These so-called “nanospray” embodiments form droplets which are only about 100 to 200 nanometers in diameter. The spray jet can then be pointed directly into the entrance of the transfer capillary from a distance of about two millimeters. In this case, no charging of surfaces inside the vacuum system takes place. The droplets appear to evaporate fully on their way through the transfer capillary.
With the matrix-assisted laser desorption and ionization at atmospheric pressure method (AP-MALDI), only recently commercially introduced, the ionization cloud is produced by laser light bombardment from a pulsed evaporated sample. The ionization cloud initially only consists of a matrix vapor with few analyte molecules blown into the gas phase. Only a tiny proportion of the molecules, of the order of a hundredth of a percent or less, are ionized. The cloud rapidly mixes with the surrounding gas. Here, the matrix ions does not necessarily have to perform the ionization, as is necessary in a vacuum; other processes have been disclosed which separate the ionization from the desorption (J. Franzen and C. Köster, U.S. Pat. No. 5,663,561). With this method of ionization, in principle, no droplets have to be dried but it is also desirable for a high proportion of the ions, as in the case of the spray method, to be transferred to the vacuum of the mass spectrometer. It is also desirable to ionize more analyte molecules than before.
In principle, any type of mass spectrometer can be used to analyze the ions from the ionization cloud, provided it is possible to make the ionization process sufficiently continuous. Both conventional sector-field spectrometers and quadrupole mass spectrometers can be considered and both types can be used in the form of tandem mass spectrometers to carry out MS/MS analyses.
Time-of-flight mass spectrometers require the orthogonally injected ion beam to be outpulsed into the drift tube. These OTOF mass spectrometers can be used to advantage, because the yield of charged ions to be measured is higher for these than it is for sector-field or quadrupole spectrometers, which act as a filter for a single measured ion mass only.
Storage mass spectrometers such as quadrupole ion-trap or ion-cyclotron resonance instruments are particularly advantageous both for continuous and non-continuous ion generation. These instruments are particularly suitable for scanning daughter or granddaughter ion spectra since they can be used to select and fragment individual ion species in several known ways.
Although the electrospray ion sources in particular have ecperienced many years of development, and there are numerous commercially manufactured ion sources on the market, their development is by no means complete yet. With the previous developments, the emphasis was predominantly on stable operation and not on the highest ion yield. Measured inside the vacuum, a good electrospay is capable of delivering a maximum of 100,000 ions per second. If the spray beam is guided directly at the capillary entrance then, for a brief moment, it is possible to observe an ion beam with a current which is ten to thirty times higher. This drops off within a short time, however, and soon stops altogether. If the capillary entrance is cleaned, the ion beam is raised again, although not to the maximum value, but then drops off just as quickly. Signs of the metal surfaces in the area of the entrance and even in the vacuum area after the transfer capillary becoming charged have been observed. The impact of ions on a clean metal surface only leads to surface charging when there is an extreme predominance of heavy ions. For this reason, either finest drops must play a rôle which, in spite of the hot gas in the counterflow, can reach the metal surface, where they can condense and remain in spite of the high temperature of the surface. This process may also involve polymerization of the liquids due to the impact of reactive ions. Or, surfaces such as those around the entrance of the transfer capillary are covered to a very large extent with analyte ions. The surfaces can only become charged if the coatings consist of at least ten monomolecular layers since the charges can be conducted away from thinner coatings. A coating such as this must contain at least 10 picomol of analyte substance per square millimeter. This value can only be reached, within a period of hours, if the ion guidance is extremely unfavorable and if the majority of the ions are not sucked into the entrance of the transfer capillary.
In any case, the large ion current, which can be achieved for a brief time, indicates that, in principle, very many more ions can reach the vacuum of the mass spectrometer than is the case for the ion spray sources which are in use nowadays. It is, therefore, not the much-feared space charge limit in the transfer capillary which restricts the flow of ions. A method has also been disclosed showing how the ions in the transfer capillary can be hydrodynamically focused in order to avoid ion losses (J. Franzen, U.S. Pat. No. 5,736,740).